More than three decades ago, Hans Selye developed the stress-response model of aging. He described a progressive degeneration in which the organism ultimately fails to recover from the physical, mental and emotional effects of stress. To this day, Selye's model is accepted as one of the lynch pins of aging research. Brain degeneration, once thought to be an "inevitable" consequence of aging appears to be directly related to stress hormone levels. Dr. Robert Sapolsky, a MacArthur Fellow and Stanford biologist, found strong evidence that chronic emotional and oxidative stress, and not aging per se, causes brain degeneration. Current research indicates stress-related factors in Alzheimer's disease and other disorders.
Stress, whether physical, emotional or biochemical, produces alterations in hormone levels. Specifically, stress causes an increase in blood levels of corticosteroids, such as cortisol and a decrease in androgenic steroids, such as testosterone. Elevated levels of stress hormones have been determined to exert powerfully negative effects on the immune system, and may account significantly for age-related increases in auto-immune disorders such as lupus, multiple sclerosis, rheumatoid arthritis, fibromyalgia and chronic fatigue syndrome.
The terms anabolic and catabolic are commonly used in physiology to denote the build-up and break-down functions of both cells and tissues or entire systems. The Anabolic/Catabolic Index (ACI) test measures and reports the ratio of anabolic and catabolic activity in humans. It is a global/snapshot view of repair and rebuild (anabolic) activity versus waste activity (catabolic). It is important not to confuse this analysis with cellular catabolism, which refers to the biochemical reduction of compounds to create energy. For example, the breakdown of complex carbohydrates to yield glucose is a catabolic process. The ACI test, on the other hand, assesses the building up or tearing down of structural tissues, such as, muscle and other connective tissue.
Stress produces alterations in the anabolic/catabolic index, and may be influenced by factors favoring anabolic (recovery) activity. Simply measuring blood levels of cortisol (17-OHCS), also referred to as hydrocortisone, has been considered as a biomarker for catabolic activity. However, this measurement does not provide sufficient information to be relied on for this purpose. Cortisol, is the major corticosteroids hormone secreted by the adrenal cortex. The clinical usefulness of cortisol measurement is limited by the fact that stress induces a biphasic response in the secretion of cortisol. At some point of adrenal exhaustion, cortisol levels decrease even though the individual continues to deteriorate.
Various researchers have used the ratio of serum testosterone and cortisol as an anabolic/catabolic index (Adlercreutz et al. (1986) lnt. J. Sports Med. 7:27-28; Obminski and Stupnicki (1997) J. Sports Med. Phys. Fitness 37:50-55), while others use the ratio of non-SHBG-bound testosterone (NST) to cortisol (Tegclman et al. (1992) Int. J. Sports Med. 13:424-430) to measure the ACI. Recent research, however, suggests that neither testosterone nor NST is an ideal biomarker for catabolic activity. The measurement of testosterone produces inconsistent results in females (Vervoom et al. (1992) Eur. J. Appl. Physiol. 64:14-21), and is not reflective of alterations in IGF and IGFBP which contribute to the catabolic state. (Gelato and Frost (1997) Endocrine 7:81-85). IGF-I has also been used as an anabolic biomarker (Van Wyk (1984) in Hlormonal Proteins and Peptides, vol. XII, (Li, C. H. ed.) Academic Press, N.Y., pp 82-125), but turned out to be unreliable due to its hypersensitivity to shout-term changes in caloric intake, sleep and exercise. (Clemmons and Van Wyk (1984) J. Clin. Endocrinol. Metab. 13:113-143).
Urinary 17 ketosteroids (17-KS) consist primarily of 17 KS sulfate conjugates (17 KS-S) and 17 KS glucuronides (17 KS-G), the latter derived from cortisol, DHEA (dehydroepiandrosterone) and testosterone. (See FIG. 1). The measurement of steroids in human serum and urine specimens has been utilized as a clinical indicator of adrenal function (Lobo et al. (1981) Obstet Gynecol, 57:69-73), androgen abuse (Dehennin et al. (1998) Steroids 63:80) and as a indicator of general health (Field et al. (1994) J. Clin. Endocrinol. Metab. 79:1310). Researchers at Hokkaido University in Japan have recently proposed that a more comprehensive biomarker for anabolic activity is the measurement of total urinary 17 ketosteroid sulfate conjugates (17 KS-S). (Nishikaze and Furuya (1998) J. UOEH 20:273). This biomarker, which is comprised of four different hormone metabolites (DHEA-S, androsterone, etiocholanolone and epiandrosterone, see FIG. 1) reflects the full range of anabolic repair/rebuild activity in muscles, organs, connective tissue, immune and nervous systems. The amount of 17-KS-S from 24-hour secretion corrected with creatinine has been found to parallel the recovery from infection, illness, injury and psychosocial stress. (Van Wyk (1984) in Hormonal Proteins and Peptides, vol. XII, (Li, C. H. ed.) Academic Press, N.Y., pp 82-125; Furuya et al. (1998) Jpn. J. Clin. Pathol 46:529-537).
Dehydroepiandrosterone (DlIEA), also referred to as, dehydio-3-isoandrosterone or 3-.beta.-hydroxyandrost-5-ene-7-one, is a weakly androgenic steroid secreted primarily by the adrenal gland. It is one of the principal components of urinary 17-ketosteroids. DHEA(S) hydrosteroid sulfatases convert DHEA to DHEA-S. DHEA and DHEA-S levels correlate to stress, central nervous system function, immunological function, cardiovascular disorders and insulin sensitivity. (Kroboth et al. (1999) J. Clin. Pharmacol. 39:327).
The results of steroid profiling confirm that the measurement of total ketosteroids in the urinary steroid sulfate fraction represent only metabolites of anabolic activity. Therefore, factoring out the glucuronides gives a more precise indication of pure anabolic activity, which provides a sensitive and accurate picture of repair and rebuild activity in both women and men.
One method used to measure 17-KS-S levels involves a calorimetric assay (Zimmerman chromogen reaction). Although this method is widely used, it does not differentiate between ketosteroids nor between free and sulfate or glucuronide fractions. Additionally, the Zimmerman reaction also measures a significant amount of the degradation and conversion products resulting from peripheral glucocorticoid and testosterone metabolism; that is compounds having a C(O)CH.sub.2 group not only at C17, but also at C3, C6 and C20. To obtain a true anabolic biomarker, glucocorticoid and other metabolites must be removed.
A second method currently used to measure 17-KS-S levels involves chromatographic separation followed by colorimetric reaction, which is outlined below and described in Example 1. (Setchell et at. (1976) J. Steroid Biochemistry 7:615-629). ##STR1##
Although this method is specific for 17 KS-S, it is a time consuming process (involving two chromatographic separations) and it is expensive. This has been used as a research rather than a diagnostic method. More recently, mass spectroscopic detection conjugated with gas chromatography (GC) or high pressure liquid chromatography (HPLC) has been reported for quantification of 17-detosteroid conjugates (Shackleton et al. (1990) Steroids 55:472) and estrogen sulfates (Zhang and lienion (1999) Anal. Chem. 71:3955).
Urinary 17 KS-S levels are high in young, healthy individuals, decrease with aging and failing health, clearly decline with advancing disease and reach very low levels in severe disease and old age. This biomarker, therefore, provides a snapshot view of the body's regenerative capacity and "biological age." Although there is no established definition of "biological age," this term is frequently used to described the functional status of the human body as opposed to its chronological age. Current methods used to determine biological age include, measurement of skin thickness, strength, stamina, body composition, reaction time, vision, hearing, blood and neurological tests. All of these tests have one common feature, in that they measure aspects of anabolic and catabolic activity not as an active kinetic process, but rather as a clinical endpoint. To date, there has been no quantified correlation between 17 KS-S levels and biological age.